Cultivation method for efficient in-vitro rapid propagation of sweet potato virus-free seedlings

Abstract

The invention discloses a cultivation method for efficient in-vitro rapid propagation of sweet potato virus-free seedlings, and belongs to the technical field of plant breeding. The method comprises the following steps: by taking new buds germinated from excellent plant tubers of a bred sweet potato variety as a material source, performing stem efficient adventitious bud induction and sterile system establishment, stem tip stripping and bud induction, adventitious bud proliferation and elongation synchronous culture, regenerated plant detoxification effect detection, ex-vitro adventitious root induction and domestication transplanting synchronous culture and the like. And finally, efficient propagation of the detoxified sweet potato seedlings is realized. The method provides key technical support for detoxification of sweet potato excellent strain seedlings and industrialized production of detoxified seedlings, and can meet the annual requirements of industrialized development on high-quality seedlings.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant breeding, and particularly relates to a high-efficiency in-vitro rapid propagation cultivation method for virus-free sweet potato seedlings. BACKGROUND

[0002] As the seventh largest food crop in the world, sweet potato plays an irreplaceable strategic role in ensuring global food security, providing nutritional supply, and creating economic value. Sweet potato is rich in dietary fiber, vitamins, minerals, and functional polysaccharides, and has multiple uses such as food security reserve, healthy food processing, and feed production. It is a typical "food, feed, and fiber" crop with unique advantages in optimizing agricultural industry structure. As the world's largest sweet potato producer and consumer, China has long been leading in terms of planting area and total production. The sweet potato industry has become a key component in ensuring the diversity of domestic food supply and boosting agricultural economic growth.

[0003] However, the current development of the sweet potato industry faces two major bottlenecks, highlighting the necessity and urgency of research and development of seedling virus elimination and efficient propagation technology. On the one hand, sweet potato virus disease, a global disease, can cause plant dwarfing, leaf curling, and root deformity, resulting in 10%-50% yield loss and even complete loss in severe cases. The virus can be transmitted by aphids, whiteflies, and other vectors, and accumulates from generation to generation through vegetative propagation materials, becoming one of the key bottlenecks restricting the quality improvement and industrial scale development of sweet potato. On the other hand, sweet potato is mainly propagated using traditional vegetative propagation methods, relying on tubers and stems. Although these methods are simple and have low initial costs, they have low propagation efficiency, high risk of disease transmission, and seedling quality is affected by environment and management, with significant differences in uniformity and stress resistance, which seriously affects yield and quality uniformity in the field. Therefore, the development of efficient, low-cost, and industrialized sweet potato virus elimination and rapid propagation systems using modern biotechnology is of great practical significance for improving seedling quality, ensuring supply stability, promoting the upgrade of the sweet potato industry from "quantity-based" to "quality and efficiency-based", and ensuring national food security and promoting agricultural efficiency and farmers' income.

[0004] Plant stem tips have very low virus content and are the core material for virus-free seedling production. Stem tip virus elimination technology and rapid propagation technology based on virus-free materials have become a core production approach to replace traditional propagation methods. Currently, sweet potato stem tip virus elimination and virus-free seedling rapid propagation technology has made some progress, but traditional sweet potato virus-free stem tips have low regeneration efficiency, low regeneration coefficient, and complex operation process, and virus elimination is not complete. In addition, current sweet potato virus-free seedling propagation relies solely on using axillary bud stem segments as explants and relies on the germination of axillary buds carried by the stem segments for propagation, resulting in low propagation coefficient. Therefore, it is urgent to develop efficient stem tip virus elimination and virus-free seedling rapid propagation technology systems. SUMMARY

[0005] The technical problem to be solved by this invention is how to solve the problem of low propagation coefficient and poor effect of axillary bud germination and propagation carried by sweet potato stem segments.

[0006] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes a method for efficient in vitro propagation of virus-free sweet potato seedlings, comprising the following steps: (1) Induction of adventitious buds in stem segments and establishment of a sterile system: Stem segments with new shoots from sweet potato tubers with buds were cut, rinsed, disinfected, and inoculated into MS medium 1 for culture to induce adventitious buds; (2) Shoot tip stripping and induction culture: The adventitious buds obtained in (1) were separated from the stem tip and inoculated into the second MS medium for culture, and adventitious buds were induced again. (3) Simultaneous culture of adventitious bud proliferation and elongation: After removing the callus tissue at the base of the stem tip of the adventitious buds obtained in (2), they were transferred to the third MS medium for culture until elongated adventitious buds were produced. (4) Detection of virus detoxification effect in regenerated plants: Leaves from the elongated adventitious buds obtained in (3) were used to detect sweet potato chlorosis dwarf virus (SPCSV) and sweet potato feather mottle virus (SPFMV) using the loop-mediated isothermal amplification (LAMP) method; virus-free tissue culture seedlings were retained, and stem segments with buds were inoculated into the first MS medium for culture to induce adventitious buds; (5) Simultaneous culture of adventitious root induction and acclimatization transplantation outside the bottle The adventitious buds obtained in (4) are separated and cut into stem segments with buds. They are rinsed, disinfected, and the base of the stem segments is treated in IBA solution before being planted in seedling trays and cultivated until seedlings are formed.

[0007] IBA: Chinese name is indolebutyric acid, CAS Registry Number 133-32-4.

[0008] Preferably, in step (1), the new shoots of the sweet potato tubers are robust new shoots sprouting from tubers of selected superior sweet potato strains. More preferably, the sweet potato variety is Pushu 32.

[0009] Preferably, in step (1), the length of the stem segment is 2-3 cm.

[0010] Preferably, in step (1), the rinsing specifically involves rinsing with water for 10 to 20 minutes.

[0011] Preferably, in step (1), the disinfection specifically involves wiping the surface of the stem segment with alcohol, then immersing it in alcohol for disinfection, and finally disinfecting it with mercuric chloride.

[0012] Preferably, in step (1), the first MS medium is an MS medium supplemented with 6-8 g / L agar, 25-35 g / L sucrose, and 0.5-3.0 mg / L meta-Topolin. More preferably, it is 6.8 g / L agar, 30 g / L sucrose, and 2.0 mg / L meta-Topolin.

[0013] meta-Topolin: Its Chinese name is 3-[(9H-purine-6-ylamino)methyl]phenol, and its CAS number is 75737-38-1.

[0014] Preferably, in step (2), the size of the peeled stem tip is 0.1~1.0 mm.

[0015] Preferably, in step (2), the second MS medium is an MS medium supplemented with 6-8 g / L agar, 25-35 g / L sucrose, and 0.1-1.0 mg / L meta-Topolin. More preferably, it is 6.8 g / L agar, 30 g / L sucrose, and 0.5 mg / L meta-Topolin.

[0016] Preferably, in step (3), the third MS medium is an MS medium supplemented with 6-8 g / L agar, 25-35 g / L sucrose, and 0.2-2.5 mg / L meta-Topolin. More preferably, it is 6.8 g / L agar, 30 g / L sucrose, and 2 mg / L meta-Topolin.

[0017] Preferably, in step (5), the length of the stem segment with buds is 1.0~4.0cm.

[0018] Preferably, in step (5), the disinfection specifically involves placing the stem segments in a 0.05%~0.1% (w / v) potassium permanganate solution for 10~20 min.

[0019] Preferably, in step (5), the concentration of the IBA solution is 500~2000 mg / L.

[0020] Preferably, in step (5), the treatment time in the IBA solution is 10~40 s.

[0021] Preferably, in step (5), the seedling tray contains a mixed nutrient substrate; the mixed nutrient substrate is obtained by mixing nutrient soil and vermiculite in a mass ratio of 3:1.

[0022] Preferably, in steps (1) to (5), the cultivation conditions are: 23~27℃, and the light intensity is 40~50µmol / m². -2 s-1 The photoperiod is 12-16 / 12-8 h (light / dark); more preferably 25℃, 45µmol m -2 s -1 , 14 / 10h.

[0023] The beneficial effects of this invention are as follows: This invention proposes a method for the efficient in vitro rapid propagation of virus-free sweet potato seedlings, which has the following outstanding advantages: Firstly, this invention uses the newly sprouted tubers of selected superior strains as initial materials. It forms a closed loop through steps such as inducing adventitious buds from stem segments, virus-free regeneration from shoot tips, detecting the virus-free effect of regenerated buds, and simultaneously cultivating adventitious roots of regenerated plants and transplanting them, ensuring the virus-free detoxification of the final high-quality seedlings and the efficient and low-cost propagation of virus-free plants.

[0024] Secondly, this invention uses the novel plant growth regulator meta-Topolin to induce, proliferate, and elongate the adventitious buds of sweet potato stem segments and shoot tips, significantly improving the detoxification efficiency and propagation coefficient of sweet potato plants.

[0025] Thirdly, based on the obtained virus-free plants, an ex-bottle rooting mode using 500-2000 mg / L IBA for a short time (10-60s) combined with mixed substrate transplanting is adopted to replace the traditional in-bottle rooting culture medium. This eliminates the need for culture medium preparation and management during the in-bottle rooting stage, reduces seedling costs, simplifies seedling production steps, and can improve the rooting rate and seedling rate of tissue culture elongated shoots. Rooting and seedling formation can be achieved in one week, with an adventitious root induction rate as high as 95.6%.

[0026] Fourth, this method provides key technical support for the virus-free seedling production of superior sweet potato lines and the industrialized production of virus-free seedlings, which can meet the year-round demand for high-quality seedlings in industrial development. This method can significantly improve the propagation coefficient and survival rate of virus-free seedlings after transplanting, realizing rapid and efficient industrialized propagation of virus-free seedlings of superior sweet potato lines.

[0027] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0028] Figure 1 This is a diagram of stem segments from superior sweet potato lines cultured in MS medium for 2 weeks in Example 1; Figure 2 The image shows a stem segment of a sterile sweet potato seedling cultured for 2 weeks in MS medium supplemented with 2.0 mg / L meta-Topolin, as described in Example 1. Figure 3 The image shows a stem segment of a sterile sweet potato seedling cultured for 4 weeks in MS medium supplemented with meta-Topolin 2.0 mg / L, as described in Example 1. Figure 4 This is a diagram of the 0.3 mm shoot apical meristem that was peeled off in Example 1; Figure 5 This is a diagram of the shoot tips cultured on the bud induction medium for 5 weeks in Example 1; Figure 6 This is an image of the shoot cluster obtained after the shoot tip was cultured on the shoot induction medium for 7 weeks in Example 1; Figure 7 This is an image of the adventitious shoot clusters obtained from the simultaneous proliferation and elongation culture of Example 1 over 4 weeks; Figure 8 This is a diagram showing the elongation of buds from sweet potato stem segments after one week of cultivation in a nutrient substrate, as described in Example 1. Figure 9 This image shows a robust, virus-free, regenerated sweet potato plant obtained after culturing in a nutrient substrate for 3 weeks, as shown in Example 1. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0030] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0031] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0032] Example 1: A method for efficient in vitro rapid propagation of virus-free sweet potato seedlings includes the following steps: (1) Induction of adventitious buds from stem segments and establishment of a sterile system: Healthy new shoots sprouting from tubers of the selected sweet potato variety "Pushu 32" were used as explants. Stem segments 2-3 cm long with buds were cut as materials. After rinsing with tap water for 20 min, the surface was wiped with 75% alcohol, then placed on a sterile operating table and washed with sterile water 6 times, followed by sterilization with 75% ethanol for 45 s. Then, after sterilization with 0.1% mercuric chloride for 3 min, the segments were washed with sterile water 6 times. The surface of the stem segments was dried with filter paper and cut into 0.8-1.5 cm segments with buds. These segments were inoculated into MS medium supplemented with 6.8 g / L agar, 30 g / L sucrose, and 2.0 mg / L meta-Topolin at 25℃ and a light intensity of 45 μmol·m⁻¹. -2 ·s -1 Shoot induction and the establishment of a sterile system were conducted in a greenhouse with a photoperiod of 14 h / 10 h. The results showed that after two weeks of culture, stem segments inoculated into a medium supplemented with 2.0 mg / L meta-Topolin could induce adventitious shoots, with an induction rate as high as 100%, and an average of 3.3 adventitious shoots per explant. Figure 2 After two more weeks of cultivation, adventitious bud clusters with an average plant height of 3-4 cm were obtained. Figure 3 ).

[0033] (2) Shoot tip stripping and induction culture: Using the sweet potato adventitious shoots obtained in step (1) as materials, shoot tips with a size of 0.3~0.5 mm were stripped under a stereomicroscope on a sterile operating table as explants. Figure 4 The buds were inoculated into MS medium supplemented with 0.5 mg / L meta-Topolin, 6.8 g / L agar, and 30 g / L sucrose and induced to develop shoots in the aforementioned greenhouse. After 5 weeks of culture, buds were induced ( Figure 5 After two more weeks of cultivation, adventitious bud clusters were induced, with an induction rate of 93.6%, and an average of 3.2 adventitious buds per shoot tip. Figure 6 ).

[0034] (3) After removing the basal callus tissue from the shoot tip explants induced with adventitious buds in step (2), the explants were transferred to MS medium supplemented with 2.0 mg / L meta-Topolin, 6.8 g / L agar and 30 g / L sucrose for simultaneous culture of adventitious bud proliferation and elongation in a greenhouse. After 4 weeks of light culture, the proliferation and elongation of sweet potato adventitious buds were achieved. On average, each explant produced 5.1 elongated adventitious buds, and the average height of the adventitious buds was 4.2 cm.

[0035] (4) Randomly select leaves of sweet potato leaves with elongated adventitious buds obtained in step (3) and use the LAMP method to detect sweet potato chlorosis dwarf virus (SPCSV) and sweet potato feather mottle virus (SPFMV). The results showed that the virus elimination rate was over 92%. Retain virus-free tissue culture seedlings and use stem segments of virus-free seedlings as explant materials. Inoculate them into the adventitious bud induction medium with 2.0 mg / L meta-Topolin added in step (1) for adventitious bud induction and subsequent propagation.

[0036] (5) After separating the virus-free, elongated adventitious buds of sweet potato obtained in step (4) from the bud cluster and rinsing them with running water, place them in a 0.1% potassium permanganate solution for 10 minutes for disinfection, then dry the surface moisture. After treating the base of the stem segment in a 1000 mg / L IBA solution for 10 seconds, transplant them into seedling trays containing a mixed nutrient substrate (mass ratio of nutrient soil to vermiculite = 3:1) treated with a 0.15% potassium permanganate solution. Induce adventitious roots and cultivate seedlings in a greenhouse, spraying with tap water once a week to ensure the substrate is thoroughly soaked. After one week of cultivation, a large number of adventitious roots were induced at the base of the stem segment ( Figure 8 After 3 weeks of cultivation, robust virus-free regenerated sweet potato plants were obtained. Figure 9 ).

[0037] Example 2: The difference between this embodiment and Embodiment 1 is that: In step (1), the inoculum was placed in MS medium supplemented with 6 g / L agar, 25 g / L sucrose, and 0.5 mg / L meta-Topolin at a temperature of 27°C and a light intensity of 40 μmol·m⁻¹. -2 ·s -1 The photoperiod is 16h / 8h; In step (2), the inoculum was inoculated into MS medium supplemented with 1.0 mg / L meta-Topolin, 8 g / L agar and 35 g / L sucrose; In step (3), the cells were transferred to MS medium supplemented with 0.2 mg / L meta-Topolin, 6 g / L agar and 25 g / L sucrose; In step (5), the sample was treated with 500 mg / L IBA solution for 40 seconds; The rest is the same as in Example 1.

[0038] Example 3: The difference between this embodiment and Embodiment 1 is that: In step (1), the inoculum was placed in MS medium supplemented with 8 g / L agar, 35 g / L sucrose, and 3.0 mg / L meta-Topolin at a temperature of 23 °C and a light intensity of 50 μmol·m⁻¹. -2 ·s-1 The photoperiod is 12h / 12h; In step (2), the inoculum was inoculated into MS medium supplemented with 0.1 mg / L meta-Topolin, 6 g / L agar and 25 g / L sucrose; In step (3), the cells were transferred to MS medium supplemented with 2.5 mg / L meta-Topolin, 8 g / L agar and 35 g / L sucrose; In step (5), the sample is treated in a 2000 mg / L IBA solution for 10 seconds; The rest is the same as in Example 1.

[0039] Example 4: This embodiment tested the effects of different plant growth regulators on the induction of adventitious buds in stem segments of virus-free sweet potato seedlings. The specific steps are as follows: Using stem segments (approximately 0.5-1.0 cm long) with axillary buds from virus-free sterile sweet potato seedlings obtained in Example 1 as material, these segments were inoculated into MS medium supplemented with different concentrations (0, 0.2, 0.5, 1.0, 2.0, and 3.0 mg / L) of plant growth regulators (meta-Topolin, 6-BA, and TDZ), 30 g / L sucrose, and 6.8 g / L agar. In vitro regeneration studies were conducted in the greenhouse described in Example 1. The induction of adventitious buds was observed in real-time during the culture period. After 5 weeks of light-induced culture, the average number of adventitious buds produced per explant and the average height of adventitious buds were recorded. The results are shown in Table 1, indicating that the type and concentration of plant growth regulators have a significant impact on the induction of adventitious buds from sweet potato stem segments. Among the three plant growth regulators tested, meta-Topolin was most beneficial for the induction of adventitious buds from sweet potato stem segments; within the same concentration range, the number of adventitious buds induced by meta-Topolin was significantly higher than that induced by 6-BA and TDZ. Among the different concentrations of meta-Topolin tested, its induction of adventitious buds in sweet potato stem segments showed a dose-dependent effect. As the concentration of meta-Topolin increased, the average number of adventitious buds produced per explant and the diameter of callus tissue at the base of the stem segment both showed a gradual increasing trend. Among them, 2.0 mg / L meta-Topolin induced the most adventitious buds, with an average of 4.46 adventitious buds produced per explant.

[0040] Table 1. Effects of different types and concentrations of exogenous plant growth regulators on adventitious shoot induction in sweet potato stem segments.

[0041] Note: Data are averages. Each treatment contained 30 explants and was repeated three times.

[0042] Example 5: This embodiment tested the effect of different treatment times with exogenous IBA on exogenous rooting in sweet potato tissue culture bud elongation bottles. The specific steps are as follows: After separating uniformly growing, robust sweet potato virus-free tissue culture elongation shoots from adventitious bud clusters, they were rinsed with running water and then sterilized in a 0.1% potassium permanganate solution for 10 minutes, followed by absorbing surface moisture. The bases of the sterilized sweet potato tissue culture elongation shoots were then immersed in a 1000 mg / L IBA solution for different durations (0, 10, 20, 40, and 60 s). The elongation shoot bases were then transplanted into seedling trays containing a mixed nutrient substrate (nutrient soil: vermiculite = 3:1) treated with a 0.1% potassium permanganate solution. Adventitious root induction and seedling cultivation were carried out in a greenhouse, with tap water sprayed weekly to ensure substrate saturation. The induction rate of adventitious roots, the average number of adventitious roots produced per explant, and the length of adventitious roots were regularly observed and recorded. The results are shown in Table 2. The study found that the duration of exogenous IBA treatment significantly affected the induction of adventitious roots from sweet potato tissue culture shoots and the transplant survival rate. Among the tested treatment times, the induction rate of adventitious roots initially increased and then decreased with prolonged treatment time. Treatment at the base of the stem segment for 10 seconds resulted in the best adventitious root induction effect and the highest transplant survival rate, with an induction rate of 95.8%, an average adventitious root length of 5.2 cm, an average of 14.5 adventitious roots per explant, and a 100% transplant survival rate.

[0043] Table 2. Effects of exogenous IBA treatment time on the survival rate of sweet potato tissue culture seedlings after transplanting.

[0044] Note: Data are averages. Each treatment contained 30 explants and was repeated three times.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cultivation method for efficient in vitro rapid propagation of virus-free sweet potato seedlings, characterized in that, The method comprises the following steps: (1) Induction of adventitious buds of stem segments and establishment of a sterile system: Cut the stem segments of sweet potato strains with bud points, wash and disinfect them, inoculate them in the first MS medium, and culture them to induce adventitious buds; (2) Dissection of stem tips and induction culture: Dissect the stem tips of the adventitious buds obtained in (1), inoculate them in the second MS medium, and culture them to induce adventitious buds again; (3) Synchronous culture of proliferation and elongation of adventitious buds: After the stem tips of the adventitious buds obtained in (2) are cut to remove the basal callus, they are transferred to the third MS medium for culture until elongated adventitious buds are generated; (4) Detection of detoxification effect of regenerated plants: Take the leaves of the elongated adventitious buds obtained in (3), and use loop-mediated isothermal amplification to detect sweet potato chlorosis stunt virus and sweet potato feathery mottle virus; retain the virus-free tissue culture seedlings, inoculate the stem segments with bud points in the first MS medium to induce adventitious buds; (5) Synchronous culture of adventitious root induction and acclimatization and transplanting outside the bottle Separate the adventitious buds obtained in (4) into stem segments with bud points, wash and disinfect them, treat the base of the stem segments in an IBA solution, and then plant them in a seedling tray for culture until seedlings are generated.

2. The breeding method according to claim 1, characterized by, In step (1), the length of the stem segments is 2-3 cm.

3. The breeding method according to claim 1, characterized by, In step (1), the first MS medium is MS medium added with 6-8 g / L agar, 25-35 g / L sucrose and 0.5-3.0 mg / L meta-Topolin.

4. The breeding method according to claim 1, characterized by, In step (2), the size of the dissection stem tips is 0.1-1.0 mm.

5. The breeding method according to claim 1, characterized by, In step (2), the second MS medium is MS medium added with 6-8 g / L agar, 25-35 g / L sucrose and 0.1-1.0 mg / L meta-Topolin.

6. The breeding method according to claim 1, characterized by, In step (3), the third MS medium is MS medium added with 6-8 g / L agar, 25-35 g / L sucrose and 0.2-2.5 mg / L meta-Topolin.

7. The breeding method according to claim 1, characterized by, In step (5), the length of the stem segments with bud points is 1.0-4.0 cm.

8. The breeding method according to claim 1, characterized by, In step (5), the concentration of the IBA solution is 500-2000 mg / L; the treatment time in the IBA solution is 10-40 s.

9. The breeding method according to claim 1, characterized by, In step (5), the seedling tray contains mixed nutrient substrate; the mixed nutrient substrate is obtained by mixing nutrient soil and vermiculite at a mass ratio of 3:

1.

10. The breeding method according to claim 1, characterized by, In steps (1)-(5), the culture conditions are as follows: 23-27℃, light intensity of 40-50 µmol m -2 s -1 , photoperiod of 12-16 / 12-8 h (light / dark).

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